The invention relates in general to the isolation of certain types of animal cells and in particular to the isolation of specific types of cells found in the eye.
FIG. 8 is a side sectional view of a human eye 28. The eye 28 has a lens 30 and a cornea 32. FIG. 9 is an enlarged sectional view of the cornea 32. Mammalian corneal endothelial cells (CECs) 34 are a single layer of cells located on the posterior side of the cornea 32, facing the anterior chamber. The outermost layer of the cornea 34 is the epithelium 36. CECs 34 allow nutrients from the anterior chamber to pass into the cornea 32. CECs 34 pump water out of the cornea 32 and into the anterior chamber. CECs 34 in the intact adult human eye have limited growth potential. CECs demonstrate highly limited growth potential in vivo. A reduction in CEC number is normally observed during aging, but this is usually accommodated for by an increase in average cell diameter.
If a critical number of CECs are lost due to disease or injury, this compensatory response is no longer sufficient, and the endothelial barrier is breached. Subsequent corneal edema results in significant inflammation, epithelial bullae, and limbal stem cell deficiency. Together these complications can eventually lead to corneal opacity and total vision loss. The best characterized examples of CEC loss include Fuch's dystrophy (a genetically-based degenerative disease of the corneal endothelium), aphakic/pseudophakic bullous keratopathy (PBK, resulting from endothelial cell injury incurred during cataract surgery) and mustard gas keratopathy (MGK, which occurs following ocular exposure to the chemical warfare agent sulfur mustard). Surgical intervention by corneal transplant is the only currently available option for patients with critical CEC loss. However, transplantation is often unavailable due to the limited supply of fresh corneas suitable for transplantation. Patients that do receive donated tissue face the possibility of transplant rejection.
A known process for isolating CECs is called Descemet's stripping. This procedure involves the use of a sharp, bladed instrument known as a trephine. The trephine is used to scrape CECs 34 away from the underlying basement membrane 38 (Descemet's Membrane). This method can frequently result in the co-isolation of corneal stromal cells 40 (keratocytes) located beneath Descemet's Membrane 38. Keratocytes 40 have a very high proliferative potential. Therefore, CEC isolation by Decemet's stripping often results in the overgrowth of contaminative keratocytes 40 during cell culture expansion, rendering the expanded cells unusable for transplant or for the study of a homogenous CEC population.
A need exists for an apparatus and method for isolating pure populations of CECs from a cornea.